It is well known to employ biocatalysts, such as microorganisms that contain enzymes, for conducting chemical reactions. Nitrile hydratase enzymes are known to catalyse the hydration of nitriles directly to the corresponding amides. Typically nitrile hydratase enzymes can be produced by a variety of microorganisms, for instance microorganisms of the genus Bacillus, Bacteridium, Micrococcus, Brevibacterium, Corynebacterium, Pseudomonas, Acinetobacter, Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, Pseudonocardia and Rhodococcus. 
Many references have described the synthesis of nitrile hydratase within microorganisms. Amaud et al., Agric. Biol. Chem. 41: (11) 2183-2191 (1977) describes the characteristics of an enzyme they refer to as ‘acetonitrilase’ in Brevibacterium sp R312 which degrades acetonitrile to acetate via the amide intermediate. Asano et al., Agric. Biol. Chem. 46: (5) 1183-1189 (1982) isolated Pseudomonas chlororaphis B23 which produced nitrile hydratase to catalyse the conversion of acrylonitrile to acrylamide, generating 400 g/L acrylamide. The article by Yamada et al., Agric. Biol. Chem. 50: (11) 2859-2865 (1986) entitled, “Optimum culture conditions for production by Pseudomonas chlororaphis B23 of nitrile hydratase”, considered the optimisation of the medium components of the growth medium, including the inducer added for nitrile hydratase synthesis. Methacrylamide was found to be the best inducer for this organism. Methacrylamide was included in the culture at the start of growth.
Various strains of the Rhodococcus rhodochrous species have been found to very effectively produce nitrile hydratase enzyme.
EP-0 307 926 describes the culturing of Rhodococcus rhodochrous, specifically strain J1 in a culture medium that contains cobalt ions. A process is described for biologically producing an amide in which a nitrile is hydrated by the action of a nitrile hydratase produced by Rhodococcus rhodochrous J1, which has been cultured in the presence of cobalt ion. The use of various inducers (including crotonamide) is described for the synthesis of nitrile hydratase. In one embodiment an amide is produced in a culture medium of the microorganism in which a substrate nitrile is present. In another embodiment a substrate nitrile is added to the culture medium in which a nitrile hydratase has been accumulated to conduct the hydration reaction. There is also a description of isolating the microbial cells and supporting them in a suitable carrier, for instance by immobilisation, and then contacting them with a substrate. The nitrile hydratase can be used to hydrate nitriles into amides, and in particular the conversion of 3-cyanopyridine to nicotinamide.
EP-0 362 829 describes a method for cultivating bacteria of the species Rhodococcus rhodochrous comprising at least one of urea and cobalt ion for preparing the cells of Rhodococcus rhodochrous having nitrile hydratase activity. Specifically described is the induction of nitrile hydratase in Rhodococcus rhodochrous J1 using urea or urea derivatives which markedly increases the nitrile hydratase activity. Urea or its derivatives are added to the culture medium in one batch at a time or sequentially and cultivation occurs over 30 hours or longer, for instance up to 120 hours.
An article by Nagasawa et al., Appl. Microbiol. Biotechnol. 34: 783-788 (1991), entitled “Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1”, describes isolating J1 as an acetonitrile utilising strain which synthesises two different nitrile hydratases and a nitrilase depending upon the culture conditions used. One nitrile hydratase is induced optimally by urea and urea analogues. Urea is added at the start of the culturing process and seems to become efficient as an inducer only when the basal medium is nutrient rich. Induction of the enzyme started gradually and increased in growth until it reached a maximum after 5 days of cultivation. The activity was found to decrease on prolonged cultivation.
Rhodococcus rhodochrous J1, is also used commercially to manufacture acrylamide monomer from acrylonitrile and this process has been described by Nagasawa and Yamada Pure Appl. Chem. 67: 1241-1256 (1995).
Leonova et al., Appl. Biochem. Biotechnol. 88: 231-241 (2000) entitled, “Nitrile Hydratase of Rhodococcus”, describes the growth and synthesis of nitrile hydratase in Rhodococcus rhodochrous M8. The nitrile hydratase synthesis of this strain is induced by urea in the medium, the urea also acting as a nitrogen source for growth by this organism. Cobalt is also required for high nitrile hydratase activity. This literature paper looks at induction and metabolic effects in the main.
Leonova et al., Appl. Biochem. Biotechnol. 88: 231-241 (2000) also states that acrylamide is produced commercially in Russia using Rhodococcus rhodochrous M8. Russian patent 1731814 describes Rhodococcus rhodochrous strain M8.
Rhodococcus rhodochrous strain M33 that produces nitrile hydratase without the need of an inducer such as urea is described in U.S. Pat. No. 5,827,699. This strain of microorganism is a derivative of Rhodococcus rhodochrous M8.
The production of acrylamide monomer in particular is desirable via the biocatalytic route. In the review publication by Yamada and Kobayashi, Biosci. Biotech. Biochem. 60: (9) 1391-1400 (1996) titled “Nitrile Hydratase and its Application to Industrial Production of Acrylamide” a detailed account of the development of a biocatalytic route to acrylamide is described. Three successively better catalysts and their characteristics for acrylamide production and in particular the third generation catalyst Rhodococcus rhodochrous J1 are described in some detail.
A major disadvantage with the use of biocatalysts is the general lack of stability observed with wet microbial material during storage, transportation and use. Even with relatively stable enzymes and bacteria such as nitrile hydratases in Rhodococcal cells, the potential for spoilage before use has led to acceptance within the industry for the need to process the biocatalyst cell suspension in some way e.g. by freezing or freeze-drying of the aqueous mixture or alternatively immobilisation of the cells in some polymer matrix. In order to achieve maximum productivity from the biocatalyst it is important that the maximum biocatalytic activity is retained during its preparation and storage prior to use. In Chaplin and Bucke (1990) In: Enzyme Technology, published by Cambridge University Press, p 47 (Enzyme preparation and use) it was recognised that enzyme inactivation can be caused by heat, proteolysis, sub optimal pH, oxidation denaturants and irreversible inhibitors. A number of substances may cause a reduction in the rate of an enzymes ability to catalyse a reaction. This includes substances that are non-specific protein denaturants, such as urea.
In the presentation, Protein Stability, by Willem J H van Berkel, Wageningen University, factors that may cause deactivation or unfolding were considered and these included proteases, oxidation due to the presence of oxygen or oxygen radicals and denaturing agents causing reversible unfolding, such as urea.
Chaplin and Bucke (1990) In Enzyme Technology, published by Cambridge University Press, p 73 (Enzyme preparation and use) revealed that the key factor regarding the preservation of enzyme activity involves maintaining the conformation of the enzyme structure. Therefore it was considering important to prevent unfolding, aggregation and changes in the covalent structure. Three approaches for achieving this were considered: (1) use of additives; (2) the controlled use of covalent modification; and (3) enzyme immobilisation.
EP-B-0 243 967 describes the preservation of nitrile hydration activity of nitrilase by the addition of stabilizing compounds selected from nitriles, amides and organic acids and their salts, to a solution or suspension of the enzyme or the immobilized form of the enzyme. It clearly states in the description that while a solution or suspension of a microorganism capable of producing nitrilase that hydrates nitriles such as acrylonitrile, to produce the corresponding amides such as acrylamide may be stored at room temperature as long as the storage period is short, storage at a low temperature, especially at a temperature in the vicinity of 0° C. is preferred. It was described in EP-A-0 707 061 that addition of inorganic salts at a concentration of between 100 mM to the saturation concentration of the inorganic salts to an aqueous medium containing either a suspension of microbial cells or immobilized microbial cells, preserved the cells and enzyme activity for a prolonged period of time. This technique is described for the preservation of microbial cells that have nitrile hydratase or nitrilase activity. The addition of bicarbonate or carbonate salts to an aqueous solution of immobilized or unimmobilised microbial cells having nitrilase activity is described in U.S. Pat. No. 6,368,804. Immobilisation has frequently involved removal of the enzyme from the whole cell, before immobilising the enzyme in a matrix. However, although such immobilisation provides very good protection for the enzyme, extraction of the enzyme from the whole cell is an intricate step, which can be time-consuming, expensive and can lead to loss of enzyme. Additionally whole microbial cells can be immobilized. U.S. Pat. No. 5,567,608 provides a process of immobilising whole cell biocatalyst in a cationic copolymer which has good storage stability and prevents putrefaction.
Rhodococcus rhodochrous J1, which is used commercially to manufacture acrylamide monomer, is immobilised to (a) allow transportation and (b) to increase the longevity of the biocatalyst in use. In U.S. Pat. No. 5,567,608 the inventors state that biocatalysts are normally immobilized for use on an industrial scale, to facilitate ease of separation of the biocatalyst from the reaction product, preventing impurities from the biocatalyst eluting into the product and to assist in continuous processes and recycling of the biocatalyst. However, immobilisation is an extra processing step that requires an additional plant and the use of potentially a number of other raw materials such as alginate, carrageenan, acrylamide and other acrylate monomers, and vinyl alcohol. Thus, this is an expensive processing step.
Various other ways have been proposed for minimising the deleterious effects of enzyme inactivation in an attempt reduce the negative impact on a chemical reaction process.
It is also known to freeze dry biocatalysts in order to preserve the activity of an enzyme in storage over a prolonged period of time. Again this is a potentially expensive processing step that is normally carried out with biocatalysts prepared on a small scale. Cryopreservation in liquid nitrogen or in the vapour phase of liquid nitrogen also affords long-term storage of microbial cells but requires a constant supply of liquid nitrogen. Freezing of recovered biomass or semi-pure or pure enzymes at temperatures of <−18° C. is also known to preserve biocatalytic activity for prolonged periods of time.
Furthermore, once the cell mass is introduced to the reactor and the reaction is taking place minimisation of the loss of efficacy is critical to the operational efficiency and the process economics. Once again, immobilisation of the microbial cells into some polymer matrix is standard procedure to optimise these process parameters.
It would therefore be desirable to provide a process and a biocatalyst where these disadvantages can be overcome.